The hinge region of the hTSHR links the extracellular LRR-domain with the transmembrane domain. Recently, we identified mutations E297A, E303A, and D382A in the hinge region with a cell surface expression comparable to the wt but with a strongly reduced bovine TSH (bTSH) binding. The combined triple mutation E297A/E303A/D382A revealed a cell surface expression comparable to the wt but in comparison to the single mutants a more pronounced reduction of bTSH binding (9% of the wt), a decreased maximal cAMP signal of 43% and a 7-fold higher EC50 value than the wt when stimulated with bTSH. To investigate whether the reduced signaling effects of E297A/E303A/D382A are specific for bTSH we determined the triple mutants cAMP production and EC50 after stimulation with hTSH, which remained at the level of wt hTSHR. The major sequence difference between hTSH and bTSH is the lack of additional positively charged residues in hTSH. To verify potential complementary charge interaction of bTSH and hTSHR we next tested the hTSH analog TR1401 that differs from hTSH by four additional positively charged amino acids, which also exist in bTSH. Indeed, comparable to bTSH after stimulation with TR1401 the cAMP signal of the triple mutation was reduced to 52% of the wt and a 5.6-fold higher EC50 value was determined. Our data indicate for the first time that signal enhancing positively charged amino acids in the hTSH analog TR1401 and in bTSH are likely involved in direct electrostatic interactions with negatively charged residues E297, E303 and D382 located in the TSHR hinge region. This observation implies that the hinge region is a mediator of the superagonistic activity of this hTSH analog and bTSH. Furthermore, the identified intermolecular charged-charged interaction is the first detailed information for the orientation of TSH towards the hinge region of the GPHRs.